Abstract Impaired mitochondrial respiration plays a key role in metabolic, cardiovascular, and aging-related diseases. However, mitochondrial respirometry analysis currently requires processing of the living tissue sample within an hour after being taken from the patient. This requirement makes respirometry analysis largely unfeasible to standard clinical practice and clinical studies. We invented a new technology to assess maximal mitochondrial respiratory capacity in previously-frozen biological samples, which thus far been considered impossible by the scientific community. Side-by-side comparison confirmed that our assay accurately reflects results measured in fresh samples. Our technology thus overcomes the fundamental limitation of standard approaches that required living tissue. Moreover, our approach consists of simplified sample preparation which uses significantly less biological material. Enspire Bio will further develop this patented Frozen Mitos technology to enable respirometry measurements for clinical applications and frozen samples, which currently is not available. We will achieve our goals by Aim 1: Establishing standard protocols for oxygen consumption measurements from frozen tissue samples derived from human and mouse tissue samples. We will further improve throughput by enabling measurements of oxygen consumption from tissue homogenate instead of isolated mitochondria. Aim 2: Establishing proprietary kits and delivering those to five beta testing sites including opinion-leading academic labs, mitochondrial disease centers and a biotech and pharmaceutical company to test and provide feedback. To provide frozen sample analysis to clients that have no capacity for wet lab assays, we will develop a CRO- type service in-house where samples will be shipped to EnspireBio for analysis. Working with a set of pilot academic, clinical and industry clients will provide feedback to establish procedures and the economics of the CRO model approach for the frozen tissue respirometry.